Synthesis of branched para-nonylphenol isomers: occurrence and quantification in two commercial mixtures

Eight tertiary nonanols were synthesized via Grignard reaction and coupled by Friedel–Crafts alkylation with phenol to the corresponding nonylphenols. Six branched para-nonylphenols (NP) were obtained: 4-(3′-methyl-3′-octyl)phenol (33NP), 4-(2′-methyl-2′-octyl)phenol (22NP), 4-(2′,5′-dimethyl-2′-heptyl)phenol (252NP), 4-(2′,5′,5′-trimethyl-2′-hexyl)phenol (2552NP), 4-(2′,4′-dimethyl-2′-heptyl)phenol (242NP) and 4-(4′-ethyl-2′-methyl-2′-hexyl)phenol (4E22NP). Their structures were confirmed by GC–MS and NMR spectroscopy. These six isomers as well as the earlier synthesized 4-(3′,5′-dimethyl-3′-heptyl)phenol (353NP), 4-(3′,6′-dimethyl-3′-heptyl)phenol (363NP) and 4-(2′,6′-dimethyl-2′-heptyl)phenol (262NP) were compared with commercial NP mixtures purchased from Acros and Fluka by GC–MS (equipped with a 100 m polysiloxane column). The analyses revealed that all obtained isomers are occurring in different quantities in both commercial NP mixtures.     

Separation, synthesis and estrogenic activity of 4-nonylphenols: two sets of new diastereomeric isomers in a commercial mixture

Two sets of new diastereomeric 4-nonylphenol (NP) isomers [4-(3,4-dimethylheptan-4-yl)phenol (344NP, NP-J, L) and 4-(3,4-dimethylheptan-3-yl)phenol (343NP, NP-K, P)] were separated from a commercial NP mixture. The mixture of these diastereomers was synthesized at the same time by a single Friedel-Crafts reaction of 3,4-dimethyl-4-heptanol and phenol, and the mixture was separated into individual NPs by HPLC equipped with Hypercarb column. For the first time, in this study the stereostructure-estrogenic activity relationship of NP diastereomers was investigated. The NP isomers (NP-L and NP-P) having the beta-methyl group over the benzene ring were found to be 2-4 times more estrogenic than their diastereomers (NP-J and NP-K). In the case of the other set of diastereomer [4-(3,5-dimethylheptan-3-yl)phenol, (353NP, NP-E, G)] containing gamma-methyl group in the molecule, the gamma-methyl proton signal (delta 0.49) in the more estrogenic isomer (NP-G) also appeared in a higher field than the corresponding methyl signal (delta 0.76) of the less estrogenic isomer (NP-E).     

Estrogen equivalent concentration of 13 branched para-nonylphenols in three technical mixtures by isomer-specific determination using their synthetic standards in SIM mode with GC-MS and two new diasteromeric isomers

Thirteen isomers of branched para-nonylphenols (para-NP) in three technical mixtures were isomer-specifically determined using their synthesized standards by SIM of structurally specific ions, m/z 135, 149 or 163 with GC–MS. Of the 13 isomers, four isomers, 4-(2,4-dimethylheptan-4-yl)phenol, 4-(4-methyloctan-4-yl)phenol, 4-(3-ethyl-2-methylhexan-2-yl)phenol (3E22NP) and 4-(2,3-dimethylheptan-2-yl)phenol synthesized for their determinations were first used as standard substances. The 13 isomers in the technical mixtures individually occurred at mass percent portion of more than 2%. The total mass percent portions in the mixtures from Tokyo Chemical Industry (TCI), Aldrich, and Fluka covered with 89 ± 2%, 75 ± 4% and 77 ± 2%, respectively. The abundance of 4-(3,6-dimethylheptan-3-yl)phenol in the three mixtures was the largest with 11.1 ± 2% to 9.9 ± 0.3%, while that of 4-(2-methyloctan-2-yl)phenol was the smallest with 2.9 ± 0.3% to 3.0 ± 0.2%. Additionally, structures of four new isomers of more than 1% portion present in a technical mixture were elucidated as two pairs of diastereomeric isomers: two types of 4-(3,4-dimethylheptan-4-yl)phenol (344NP) and those of 4-(3,4-dimethylheptan-3-yl)phenol (343NP). By estrogenic assay of 13 isomers with yeast estrogen screen system, the activity of 3E22NP was the highest, while that of 4-(3-methyloctan-3-yl)phenol was the least. Their relative activities to that of 3E22NP were individually calculated. Estrogenic equivalent concentrations of the three technical mixtures were predictively evaluated. The ratio of the EEC to the conventional concentration, total mass percent portions of the 13 isomers in technical mixtures were 0.208 for TCI, 0.206 for Aldrich and 0.205 for Fluka. The predicted estrogenic activity of measured concentration of para-NP in technical mixtures was approximately 5-fold greater than the measured estrogen agonist activity.     

Transformation of 4-nonylphenol isomers during biosolids composting

4-Nonylphenol, a degradation intermediate of commercial surfactant and known endocrine disruptor, has been frequently detected at levels up to several thousand microgl(-1) in surface waters and up to several hundred mgkg(-1) (dry weight) in soil and sediment samples. Large quantities of 4-NP can be quickly sorbed by the organic rich solid phase during wastewater treatment and are concentrated in biosolids, a possible major source for 4-NP in the environment. Microbial transformation in culture studies followed different mechanisms for different 4-NP isomers, which have different estrogenic activity. Composting is a process of solid matrix transformation where biological activity is enhanced by process control. This approach has been used successfully in remediation of contaminated soils and sludges. In this study, the transformation kinetics of 4-NP and its isomers were characterized during biosolids composting. Five distinctive 4-NP isomer groups with structures relative to alpha- and beta-carbons of the alkyl chain were identified in biosolids. Composting biosolids mixed with wood shaving at a dry weight percentage ratio of 43:57 (C:N ratio of 65:1) removed 80% of the total 4-NP within two weeks. At this biosolids/wood shaving ratio (B:WS), the transformation of total 4-NP and its isomers followed second-order kinetic. Higher B:WS ratios yielded significantly slower 4-NP transformation which followed first-order kinetic. Isomers with alpha-methyl-alpha-propyl structure transformed significantly slower than those with less branched tertiary alpha-carbon and those with secondary alpha-carbon, suggesting isomer-specific degradation of 4-NP during biosolids composting.     

Estrogenicity profile and estrogenic compounds determined in river sediments by chemical analysis, ELISA and yeast assays

An effects-directed strategy was applied to bed sediments of a polluted tributary in order to isolate and identify the major estrogenic chemicals it discharges into the River Po, the principal Italian watercourse. Sediment extract was concentrated by solid phase extraction and then fractioned into 10 fractions by reversed phase high performance liquid chromatography (RP-HPLC). Estrogenic activity of whole extract and fractions were determined using a recombinant yeast assay containing the human estrogen receptor (YES). The 10 fractions and whole extract were analysed for target compounds, e.g. estrone (E1), 17beta-estradiol (E2), estriol (E3), 4-nonylphenol (NP), 4-tert-octylphenol (t-OP), bisphenol A (BPA), using both liquid chromatography-tandem mass spectrometry (LC-MS/MS) and non-competitive enzyme-linked immunosorbent assays (ELISA). The YES assay determined high estrogenic activity in whole sediment (15.6 ng/g EE2 equivalents), and positive results for fractions nr 1, 2, 6, 7 and 8. E1, E3 and NP were the main estrogenic chemicals, however, other unidentified compounds contributed to sediment estrogenicity, particularly for polar fractions nr 1 and 2. A GC-MS screening performed in scan mode identified other potential contributors such as phthalates (DBP, BBP), and OP isomers. A next sampling campaign extended to other tributaries and receiving stretches of the River Po confirmed E1, E3 and NP as major estrogenic chemicals potentially threatening other sites of the main river. In general, target compound ELISAs have been shown to be suitable tools for a rapid screening of wide areas or large numbers of environmental samples for estrogenic risk. The potential for interferences suggests however to use cautiously the concentration values obtained from some of the immunoassays.     

Biodegradation of nonylphenol polyethoxylates under Fe(III)-reducing conditions

Biodegradation behavior of nonylphenol polyethoxylates (NPEOs) under Fe(III)-reducing conditions was investigated. The study demonstrated that NPEOs could be rapidly biodegraded under Fe(III)-reducing conditions. Almost 60% of the total NPEOs were removed within three days and the maximum biodegradation rate was 34.95+/-0.84 microM d(-1). NPEOs were degraded via sequential removal of ether units under Fe(III)-reducing conditions. No nonylphenol polyethoxy-carboxylates (NPECs) were formed in this process. This ether removal process was coupled to Fe(III) reduction. Nonylphenol (NP), nonylphenol monoethoxylate (NP1EO), and nonylphenol diethoxylate (NP2EO) slightly accumulated in the anaerobic biodegradation process. The accumulation of these estrogenic metabolites led to a significant increase in the estrogenic activity during the biodegradation period. The calculated estrogenic activity reached its top on day 14 when the total concentration of these estrogenic metabolites was maximal. This is the first report of the primary biodegradation behavior of NPEOs under Fe(III)-reducing conditions. These findings are of major environmental importance in terms of the environmental behavior of NPEO contaminants in natural environment.     

Atmospheric concentrations and air-sea exchanges of nonylphenol, tertiary octylphenol and nonylphenol monoethoxylate in the North Sea

Concentrations of nonylphenol isomers (NP), tertiary octylphenol (t-OP) and nonylphenol monoethoxylate isomers (NP1EO) have been simultaneously determined in the sea water and atmosphere of the North Sea. A decreasing concentration profile appeared following the distance increasing from the coast to the central part of the North Sea. Air-sea exchanges of t-OP and NP were estimated using the two-film resistance model based upon relative air-water concentrations and experimentally derived Henry's law constant. The average of air-sea exchange fluxes was -12+/-6 ng m(-2)day(-1) for t-OP and -39+/-19 ng m(-2)day(-1) for NP, which indicates a net deposition is occurring. These results suggest that the air-sea vapour exchange is an important process that intervenes in the mass balance of alkylphenols in the North Sea.     

The environmental fate of the primary degradation products of alkylphenol ethoxylate surfactants in recycled paper sludge.

Alkylphenol ethoxylates (APEOs) are a group of non-ionic surfactants that are degraded microbially into more lipophilic degradation products with estrogenic potential, including nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), octylphenol (4-tOP) and nonylphenol (4-NP). Nonylphenol ethoxylates are used in paper recycling plants for de-inking paper and have the potential to be released into the environment through spreading of wastewater treatment sludge for soil amendment. Three samples of recycled paper sludge were collected from farmers' fields and analyzed for concentrations of NP1EO, NP2EO, 4-NP and 4-tOP. Each sample differed in the amount of time elapsed since the sludge was placed on farmers' fields. Primary degradation products of APEOs were present at low micrograms/g concentrations in the sludge samples. Differences in the concentrations of these analytes in sludge samples indicated that APEO concentrations declined by 84% over a period of 14 weeks on farmers' fields. Changes in the chromatographic patterns of acetylated 4-NP indicated that there is a group of recalcitrant nonylphenol isomers that degrades more slowly than other isomers. These data indicate that microbial degradation may reduce the risk of environmental contamination by these compounds, but more work is required to assess the toxic potential of APEOs in sludges used for soil amendment.     

LC-MS-MS analysis and occurrence of octyl- and nonylphenol, their ethoxylates and their carboxylates in Belgian and Italian textile industry, waste water treatment plant effluents and surface waters

Alkylphenols (APs), alkylphenol ethoxylates (APEOs), ethoxycarboxylate metabolites (APECs) and bisphenol A were determined in surface water using solid-phase extraction (SPE) followed by triple-quadrupole LC-MS-MS. APs were separated by LC from APECs using an acetonitrile-water-gradient without the addition of any buffer. Nonylphenol ethoxycarboxylates (NPECs) interfere in the detection of nonylphenols (NPs) when using an acidic mobile phase, because they produce the same MS-MS fragment ions (219>133 and 147). 4n-NP shows the characteristic transition 219>106; it is well suited as internal standard. Nonylphenol ethoxylates NPE(n)Os (n=1-17) were analysed separately in a second run by positive ionization using an ammonium acetate mobile phase. Textile industry discharges, the corresponding wastewater treatment plant (WWTP) effluents and the receiving rivers in Belgium and Italy were analysed. Among the substances investigated, NPE1C and NPE2O exhibited the highest concentrations in the water samples, up to 4.5 microg l(-1) NPE1C in a WWTP effluent and 3.6 microg l(-1) NPE2O in a river. The highest NP levels were found in the receiving rivers (max. 2.5 microg l(-1)). The predicted no-effect concentration (PNEC) for NP of 0.33 microg l(-1) for water species was frequently exceeded in the surface waters investigated, suggesting potential adverse effects to the aquatic environment.     

Passive dosing: an approach to control mutagen exposure in the Ames fluctuation test

By using dialysis equilibrium experiments, the sorption of a branched nonylphenol isomer [4-(1-ethyl-1,3-dimethylpentyl)-phenol] (NP111) on various humic acids (HAs) isolated from river sediments and two reference HAs was studied. The HAs were characterized by solid-state ¹³C direct polarization/magic angle spinning nuclear magnetic resonance (¹³C DP/MAS NMR) spectroscopy. Sorption isotherms of NP111 on HAs were described by a linear model. The organic carbon-normalized sorption coefficient (K_OC) ranged from 2.3 × 10³ to 1.5 × 10⁴ L kg⁻¹. Interestingly, a clear correlation between K_OC value and alkyl C content was observed, indicating that the aliphaticity of HAs markedly dominates the sorption of NP111. These new mechanistic insights about the NP111 sorption indicate that the fate of nonylphenols in soil or sediment depends not only on the content of HA, but also on its structural composition.     

Estrogenic effects of phenolic compounds on glucose-6-phosphate dehydrogenase in MCF-7 cells and uterine glutathione peroxidase in rats

In this study, we tested phenolic compounds such as bisphenol A (BPA), 4-nonylphenol (NP), 4-octylphenol (OP) and 4-propylphenol (PP) by using glucose-6-phosphate dehydrogenase (G6PD) in estrogen sensitive human breast cancer cells (MCF-7 cells) and glutathione peroxidase (GPx) in female immature Sprague-Dawley (SD) rats. This study was designed to investigate whether phenolic compounds have estrogenic effects in these useful screening methods for endocrine disruptors. We chose 6 h as the incubation period for the G6PD assay through a preliminary experiment using 17beta-estradiol (E2). Above the concentration of 1 x 10(-8) M, BPA significantly increased the G6PD activity in a concentration-dependent manner, relative to the control. NP (over the concentration of 1 x 10(-9) M) also enhanced the G6PD activity by about 1.8 times that of the control. OP produced weaker effects on G6PD than NP, and showed a tendency to increase the G6PD activity. PP did not affect the G6PD activity. These results show that BPA and NP have the effect of enhancing G6PD activities in MCF-7 cells. In the in vivo GPx assay, both BPA and E2 significantly increased the uterus wet weights and dramatically enhanced uterine GPx activities in immature female rats in a dose-dependent manner. Treatment with NP (500 mg/kg/day) increased significantly both the uterine GPx activity and the uterus wet weights in immature female rats. OP (500 mg/kg/day) also caused a significant increase in uterine GPx activity, but had no effect on the uterus wet weights. This finding indicates that the change in uterine GPx activities could be a more sensitive parameter than that of uterus wet weights in immature rats. This study implies that phenolic compounds have a weak estrogenic effects.     

Effects of waterborne exposure to 4-nonylphenol on plasma sex steroid and vitellogenin concentrations in sexually mature male carp (Cyprinus carpio)

4-Nonylphenol (NP) has been shown to elicit estrogenic responses both in vivo and in vitro. The mechanism by which NP exerts estrogenic and other endocrine-modulating effects in vivo remains unclear, however. The goal of this study was to evaluate the ability of NP to elicit estrogenic responses through indirect mechanisms of action involving the modulation of endogenous steroid hormone concentrations. Sexually mature male common carp (Cyprinus carpio) were exposed to aqueous NP concentrations ranging from <0.05 to 5.4 microg NP/l for 28-31 d. Approximately 0.5-3.5 ppm of NP was detected in pooled plasma samples or tissue samples from the carp studied. NP exposure did not significantly increase plasma concentrations of 17beta-estradiol (E2), testosterone (T) or vitellogenin (VTG). Excluding outliers, plasma E2 concentrations ranged from <175 to 700 pg E2/ml. T concentrations ranged from 940 to 24,700 pg T/ml plasma. The greatest VTG concentration detected was 52 microg/ml. One-third of the plasma samples tested contained <1 microg VTG/ml. Overall, the results of this study did not support the hypothesis that exposure to waterborne NP can modulate concentrations of steroid hormones in the plasma of sexually mature male carp. The results did, however, raise a number of questions regarding the utility of estradiol equivalent (EEQ) estimates as a means of predicting in vivo effects of estrogenic substances. Furthermore, they provide information regarding the concentrations and variability of E2, T, and VTG in the plasma of sexually mature male carp, which may aid in design and interpretation of future studies.     

Removal of estrogenic activities of bisphenol A and nonylphenol by oxidative enzymes from lignin-degrading basidiomycetes

Bisphenol A (BPA) and nonylphenol (NP) were treated with manganese peroxidase (MnP) and laccase prepared from the culture of lignin-degrading fungi. Laccase in the presence of 1-hydroxybenzotriazole (HBT), the so-called laccase-mediator system, was also applied to remove the estrogenic activity. Both chemicals disappeared in the reaction mixture within a 1-h treatment with MnP but the estrogenic activities of BPA and NP still remained 40% and 60% in the reaction mixtures after a 1-h and a 3-h treatment, respectively. Extension of the treatment time to 12 h completed the removal of estrogenic activities of BPA and NP. The laccase has less ability to remove these activities than MnP, but the laccase-HBT system was able to remove the activities in 6 h. A gel permeation chromatography (GPC) analysis revealed that main reaction products of BPA and NP may be oligomers formed by the action of enzymes. Enzymatic treatments extended to 48 h did not regenerate the estrogenic activities, suggesting that the ligninolytic enzymes are effective for the removal of the estrogenic activities of BPA and NP.     

Distribution of bisphenol-A, triclosan and n-nonylphenol in human adipose tissue, liver and brain

In this study, an analytical method was optimized for the determination of bisphenol-A (BPA), triclosan (TCS) and 4-n-nonylphenol (4n-NP), environmental contaminants with potential endocrine disruptive activities, in human tissues. The method consisted of a liquid extraction step, derivatization with pentafluorobenzoylchloride followed by a clean-up on acidified silica and detection with gas chromatography coupled with mass spectrometry (GC-ECNI/MS). Recoveries ranged between 92% and 102% with a precision below 5%. Limits of quantification ranged between 0.3-0.4 ng g⁻¹, 0.045-0.06 ng g⁻¹ and 0.003-0.004 ng g⁻¹ for BPA, TCS and 4n-NP in different tissues, respectively. The method was applied for the determination of BPA, TCS and 4n-NP in paired adipose tissue, liver and brain samples from 11 individuals. BPA could be detected in almost all tissues, with the highest concentrations found in adipose tissue (mean 3.78 ng g⁻¹), followed by liver (1.48 ng g⁻¹) and brain (0.91 ng g⁻¹). TCS showed the highest concentrations in liver (3.14 ng g⁻¹), followed by adipose tissue (0.61 ng g⁻¹), while it could be detected in only one brain sample. Levels of 4n-NP were much lower, mostly undetected, and therefore 4n-NP is considered of minor importance for human exposure. Despite the measurable concentrations in adipose tissue, these compounds seem to have a low bioaccumulation potential. The reported concentrations of free BPA in the various tissues are slight disagreement with pharmacokinetic models in humans and rats and therefore the possibility of external contamination with BPA during sample collection/storage cannot be ruled out.     

Developmental effects in Japanese medaka (Oryzias latipes) exposed to nonylphenol ethoxylates and their degradation products

The endocrine modulating potency of five alkylphenol compounds to fish, including nonylphenol (NP), three nonylphenol ethoxylate mixtures (NP1EO, NP4EO, NP9EO) and one nonylphenol ethoxycarboxylate (NP1EC) was assessed using in vivo tests conducted with Japanese medaka (Oryzias latipes). Medaka exposed to test materials from 1 day to 100 days post-hatch were monitored for alterations to sex ratios and secondary sex characteristics and development of gonadal intersex (i.e., testis-ova). The treatment with 100 microg l-1 NP (measured 29 microg l-1) induced gonadal intersex in over 80% of exposed males, mixed secondary sex characteristics in over 40% of exposed fish and suppression of the development of papillae on the anal fin of 100% of males. The 30 microg l-1 NP (measured 8.7 microg l-1) treatment induced gonadal intersex in only one of the 22 exposed males and mixed secondary sex characteristics in approximately 20% of the exposed fish. An elevated incidence of fish with mixed secondary sex characteristics and suppression of papillae development was also observed in the treatment with NP1EO at the highest test concentration of 300 microg l-1 (measured 105 microg l-1). There was no evidence of mixed secondary sex characteristics or gonadal intersex in treatments with the remaining test mixtures. This study confirms that NP is an estrogenic compound that could affect gonadal development in fish chronically exposed to concentrations in the range of 10 microg l-1. NP1EO is very weakly estrogenic at concentrations that are an order of magnitude higher than the lowest observed effect concentration for nonylphenol.     

Biodegradation of methoxychlor and its metabolites by the white rot fungus Stereum hirsutum related to the inactivation of estrogenic activity

The white rot fungus Stereum hirsutum was used to degrade methoxychlor [2,2,2-trichloro-1,1-bis(4-methoxyphenyl)ethane] in culture and the degraded products were extensively determined. The estrogenic activity of the degraded products of methoxychlor was examined using cell proliferation and pS2 gene expression assays in MCF-7 cells. S. hirsutum showed high resistance to methoxychlor 100 ppm, and the mycelial growth was fully completed within 8 days of incubation at 30 degrees C. Methoxychlor in liquid culture medium was gradually converted into 2,2-dichloro-1,1-bis(4-methoxyphenyl)ethane, 2,2-dichloro-1,1-bis(4-methoxyphenyl)ethylene, 2-chloro-1,1-bis(4-methoxyphenyl) ethane, 2-chloro-1,1-bis(4-methoxyphenyl) ethylene, and 1,1-bis(4-methoxyphenyl)ethylene, indicating that methoxychlor is dominantly degraded by dechlorination and dehydrogenation. MCF-7 cells were demonstrated to proliferate actively at the 10-5 M concentration of methoxychlor. However, cell proliferation was significantly inhibited by the incubation with methoxychlor culture media containing S. hirsutum. In addition, the expression level of pS2 mRNA was increased at the concentration (10-5 M) of methoxychlor. The reductive effect of S. hirsutum for methoxychlor was clear but not significant as in the proliferation assay.     

Flame retardants, surfactants and organotins in sediment and mysid shrimp of the Scheldt estuary (The Netherlands)

Sediment and mysids from the Scheldt estuary, one of the largest and most polluted estuaries in Western Europe, were analyzed for a number of contaminants that have been shown to possess endocrine-disrupting activity, i.e. organotins, polybrominated diphenyl ethers (PBDEs), hexabromocyclododecane (HBCD), tetrabromobisphenol A (TBBPA), nonylphenol ethoxylates (NPE) and transformation products, nonylphenol (NP) and nonylphenol ether carboxylates (NPEC). In addition, in vitro estrogenic and androgenic potencies of water and sediment extracts were determined. Total organotin concentrations ranged from 84 to 348 ng/g dw in sediment and 1110 to 1370 ng/g dw in mysid. Total PBDE (excluding BDE-209) concentrations ranged from 14 to 22 ng/g dw in sediment and from 1765 to 2962 ng/g lipid in mysid. High concentrations of BDE-209 (240-1650 ng/g dw) were detected in sediment and mysid (269-600 ng/g lipid). Total HBCD concentrations in sediment and mysid were 14-71 ng/g dw and 562-727 ng/g lipid, respectively. Total NPE concentrations in sediment were 1422 ng/g dw, 1222 ng/g dw for NP and 80 ng/g dw for NPEC and ranged from 430 to 1119 ng/g dw for total NPE and from 206 to 435 ng/g dw for NP in mysid. Significant estrogenic potency, as analyzed using the yeast estrogen assay, was detected in sediment and water samples from the Scheldt estuary, but no androgenic activity was found. This study is the first to report high levels of endocrine disruptors in estuarine mysids.     

Anaerobic degradation behavior of nonylphenol polyethoxylates in sludge

Anaerobic biodegradation behavior of nonylphenol polyethoxylates (NPEOs) was investigated. Results showed that terminal electron acceptors, organic matters, initial concentration, and temperature had great influence on the anaerobic biodegradation of NPEOs. Anaerobic biodegradation of NPEOs could be enhanced by adding sulfate or nitrate while this process could be inhibited by adding organic matters. The maximum removal rate increased 1.24 microM d(-1) for each ten micromoles increase in initial concentration. The decrease in temperature caused a sharp decrease in the removal efficiency of NPEOs. The temperature coefficient (PHI) for the anaerobic biodegradation of NPEOs was 0.01 degrees C(-1). Nonylphenol (NP), the typical intermediate of NPEOs, could inhibit the anaerobic biodegradation of NPEOs only at high concentration. However, these environmental factors had no effect on the anaerobic biodegradation pathway of NPEOs. The accumulation of NP and short-chain NPEOs during NPEO biodegradation led to a significant increase in the estrogenic activity during the biodegradation period.